The greatest obstacle to the field of cell and tissue encapsulation/immunoisolation has historically been the lack of sufficient oxygen and nutrient transport across the polymer membranes used to encapsulate cells and tissues. The result of this insufficient gas and nutrient exchange is cell death and lowered metabolic activity. Given that most of these encapsulation devices are used in hormone replacement therapies, such as encapsulated islet cells to treat Diabetes Mellitus, a lowered metabolic activity increases many fold the tissue requirement needed to therapeutically treat the hormone deficiency and, to date, has not generated any devices with clinical applicability to treat the many millions of diabetics throughout the world. The present invention relates to a novel device within which tissue density could be increased and gas and nutrient exchange could be improved thus greatly benefit the field of immunoisolation.
After nearly thirty years of extensive research in the field of islet cell encapsulation, most of the primary contributors in this specialized area agree upon three important tenets (See References cited 1 to 19). First, that in any geometrical style of immunoisolation device, no one dimension can exceed a value of 1 mm. Second, that no device can be loaded with cells at a tissue density higher than 5-10% v/v. Third, regardless of matrix structure, diffusion of metabolites into and insulin out of such devices is often delayed and is governed by simple diffusion gradients across the distance between the cells and the periphery of the capsule. The end results of such limitations are numerous. First, in order to achieve clinical success with any such devices, the ratio of polymer to tissue that would need to be transplanted into a diabetic patient of average weight would rapidly fill the intraperitoneal cavity of a transplant recipient. Second, if any of the above outlined tenets are modified, the result is often catastrophic to the encapsulated islets. The incidence of graft failure increases dramatically, with subclinical performances in functional tests, and the evidence of device-wide cell necrosis is prevalent upon post-explant histological examination. The primary cause for these results is frequently confirmed as the hypoxic environment of isolated and further, immunoisolated islet cells. Given the increased metabolic demands of islet cells in comparison to other somatic cells, the need for improving their oxygen supply after isolation, immunoisolation, and transplantation is of the utmost importance to the possibility of such devices having clinical relevance.
Colton and another group under Per-Ola Carlsson have implemented microelectrodes to measure oxygen partial pressures within islets, in their native environment, after isolation, and post-transplant in polymer devices and free, under the kidney capsule. Both groups found similarly (See references cited 19 and 20). The oxygen partial pressures in the native pancreatic islets are the highest of any organ within the body, measuring 37-46 mmHg (compare this to the value of 13-21 mmHg for cells within the renal cortex). Upon isolation, these values fall between 14-19 mm Hg. Upon transplantation in normoglycemic animals under the kidney capsule, the values fall slightly to 9-15 mmHg. If transplanted into severely hyperglycemic animals (above 350 mg/dL) these values fall between 6-8 mmHg. When the cells are immunoisolated and, therefore, do not lend themselves easily to transplant in a vascularized region such as the kidney capsule, the oxygen values drop even further. In fact, in hyperglycemic animals, the oxygen partial pressures of islets within polymer capsules can drop to values as low as 1-2 mmHg. These nearly anoxic conditions can result in quick cell death, particularly the nearer the cell to the core of the polymer device.
The invention further relates to the inclusion of perfluorinated substances in the Biodritin polymer formulations, in order to achieve better oxygen availability for encapsulated cells or tissues. Perfluoro organic compounds are excellent solvents for oxygen, having several fold higher solubility for oxygen than water. These compounds are largely used as blood substitutes and more recently, have been used for tissue preservation after removal from animals, as well as to improve islet isolations from pancreas.
Thus, the invention especially relates to adding perfluorinated substances in the Biodritin heteropolysaccharide, gels therefrom and/or a composition, e.g., solutions having adjustable viscosity, prepared by manipulating the concentration of Biodritin heteropolysaccharide, and/or ion concentrations, e.g., calcium; and/or a gel or sol comprising the Biodritin heteropolysaccharide; the synthesis, purification and utilization of Biodritin heteropolysaccharide as a novel glycopolymer and/or heteropolysaccharide and/or gels, solutions and/or sols comprising Biodritin heteropolysaccharide. Gels are obtained by adding an inorganic ion, such as calcium ions, to Biodritin heteropolysaccharide. Sols can be obtained by treatment of a gel with a suitable agent such as a sodium salt, e.g., citrate salts or ethylene-diamine-tetraacetic (EDTA) as sodium salt. Gels can have varying viscosity by varying the amount of Biodritin heteropolysaccharide and/or inorganic ion, e.g., calcium ion; and with an amount of calcium ions, infinite gels can be obtained.
The invention further relates to adding the perfluorinated substances to a composition herein termed xe2x80x9cBiodritin polymer networkxe2x80x9d, and to methods and formulations for preparing a Biodritin polymer network. A Biodritin polymer network can comprise at least one glycosaminoglycan, e.g., chondroitin sulfate-4 and/or 6, and at least one alginic acid salt, e.g., sodium alginate, wherein at least one of the glycosaminoglycan and alginic acid salt is cross-linked. (See U.S. application Ser. No. 08/877,682, now U.S. Pat. No. 6,281,341, filed Jun. 17, 1997 and WO98/49202 with respect to xe2x80x9cBiodritinxe2x80x9d and xe2x80x9cBiodritin Polymer Networkxe2x80x9d). And, the methods and formulations for preparing a Biodritin polymer network comprises admixing the glycosaminoglycan and alginic acid salt and adding at least one cross-linking agent, e.g., an inorganic ion. For instance, a Biodritin polymer network which is believed, without necessarily wishing to be bound by any one particular theory, to be a semi-interpenetrating polymer network (s-IPN), is formed by addition of inorganic ions to a solution of a glycosaminoglycan and an alginic acid salt, e.g., calcium ions added to a solution of chondroitin sulfate-4 and/or -6 and sodium alginate (wherein sodium alginate is cross linked and has pockets of chondroitin sulfate-4 and/or -6 and/or non-cross-linked sodium alginate).
Of course, a Biodritin polymer network containing at least one Biodritin heteropolysaccharide can be obtained by covalently bonding the glycosaminoglycan and alginic acid salt present in a Biodritin polymer network, e.g., subjecting a Biodritin polymer network to a coupling reaction involving a linker molecule; for instance, forming network by addition of inorganic ions to a solution of a glycosaminoglycan and an alginic acid salt, e.g., calcium ions added to a solution of chondroitin sulfate-4 and/or -6 and sodium alginate, and subjecting network to coupling reaction, e.g., subjecting network to coupling reaction involving divinyl sulfone.
In preparing a Biodritin heteropolysaccharide or Biodritin polymer network comprising a Biodritin heteropolysaccharide, a solution of GAG, e.g., chondroitin sulfate-4 and/or -6 is linked to alginate, e.g., sodium alginate by covalent bonding via reaction with a linker molecule such as divinyl sulfone; the reaction is preferably in alkaline medium. In practicing the invention, one can employ a process to protect the calcium binding sites of alginate, also known as xe2x80x9cegg-boxxe2x80x9d sites, such that no reaction occurs to these sites during the linking reaction with the linker molecule, e.g., divinyl sulfone, and GAG, e.g., chondroitin sulfate. One can also employ a process to eliminate remaining active vinyl groups in Biodritin, after linking, by reaction with an alkanolamine such as ethanolamine, preferably at alkaline pH. And, one can employ a process to purify Biodritin after the synthesis reaction which involves removal of calcium with molecule which binds to or conjugates with calcium, e.g., EDTA, preferably followed by precipitation(s) with an alcohol such as ethanol.
The invention also relates to an additional process to prepare a Biodritin polymer network comprising perfluorinated substances and dehydrothermal cross-linking of a mixture of the polymers that form a Biodritin heteropolysaccharide. The dehydrothermal reaction can be of a dry cake-containing GAG, e.g., chondroitin sulfate and alginate, e.g., sodium alginate. The cake can be produced by freeze-drying a solution containing chondroitin sulfate and sodium alginate in proper concentrations. The calcium binding sites of alginate can be protected prior to the dehydrothermal treatment by adding calcium ions to bind to the xe2x80x9cegg-boxxe2x80x9d sites of alginate. The calcium can be removed from the xe2x80x9cegg-boxxe2x80x9d complexes by treatment of the dehydrothermal reaction product with a material which complexes or conjugates or binds with calcium, e.g., sodium EDTA. Purification can be as discussed above.
For ease of reference, Biodritin polymer networks and Biodritin heteropolysaccharides may be termed xe2x80x9cBiodritinxe2x80x9d or xe2x80x9cBiodritin productsxe2x80x9d (See also U.S. application Ser. No. 08/877,682, now U.S. Pat. No. 6,281,341 filed Jun. 17, 1997 and WO98/49202).
A primary use of Biodritin products with the perfluorinated substances and/or products derived therefrom is in applications where biocompatibility with host tissues and/or immunoisolation are issues, for example, in cell encapsulation, such as immunoisolation by microencapsulation of islets of Langerhans for diabetes control, or of other cells types or tissues. A Biodritin gel formed from Biodritin heteropolysaccharide or comprised of a Biodritin polymer network (e.g., a gel comprising at least one glycosaminoglycan, e.g., chondroitin-4 and/or -6 and at least one alginic acid salt wherein at least one of the glycosaminoglycan and alginic acid salt are cross-linked and the glycosaminoglycan and alginic acid salt are optionally (and preferably) covalently bound, preferably via a reaction involving a linker molecule such as divinyl sulfone, and the cross-linking can be performed after the covalent binding or prior thereto; generally, a Biodritin product or a product from reaction with Biodritin) can be used to form beads or microcapsules containing cells or tissues for implantation. And thus, the invention relates to such beads or microcapsules, and methods for making and using such beads or microcapsules.
Biodritin products and/or products therefrom can also be xe2x80x9cpaintedxe2x80x9d, sprayed or applied to or on top of wounds, e.g., surgical wounds or sutures, and can be used to coat surgical, monitoring, or other equipment to avoid local reaction, injury or irritation. The skilled artisan can apply such without undue experimentation considering the disclosure herein and the knowledge in the art, and typical factors such as the age, sex, weight, condition of the patient, etc. or the nature of the equipment to be coated. Biodritin products and/or products therefrom serves as a matrix material for supporting cells for culturing or other applications in which cells must maintain a suspended or non-aggregated state. Accordingly, the invention relates to xe2x80x9cpaintsxe2x80x9d, sprays, and matrices comprising Biodritin products and/or products therefrom, and to methods for making and using them.
The novel heteropolysaccharide and products therefrom, as well as the semi-interpenetrating polymer network, i.e., Biodritin heteropolysaccharide and products therefrom and the Biodritin polymer network, make use of: the biocompatibility of glycosaminoglycans (GAGs), preferably of a specific group of glycosaminoglycans, namely those which do not have defined cell binding properties, preferably chondroitin sulfates-4 and/or -6, herein referred to as CIS; and the desirable properties of algal polysaccharides and/or alginic acid, preferably in the form of a salt of alginic acid, e.g. M+ alginatexe2x88x92, wherein M is a cation, such as a metal cation, which serves as a stoichiometric counterion to balance negative charges of the alginate anion, e.g., a Group I metal, such as sodium, lithium, or other cations, e.g., organic and complex cations, e.g., ammonium. Preferably, the counterion is sodium and the salt of alginic acid is sodium alginate, which is able to form infinite gel networks in the presence of calcium ions.
The invention especially relates to an inclusion of perfluorocompounds in the polymer formulation, as emulsions produced by sonication (or other methods) as claimed and disclosed in the examples to ensure cell survival during cell or tissue encapsulation or immunoisolation for therapeutic uses.
Perfluorinated hydrocarbon derivatives are extremely dense, chemically inert, and water insoluble compounds that were developed in the early 1940""s as a part of the Manhattan Project (See references 22 and 23). Researched extensively in regards to their chemical properties for years (See references 24 to 37) they were selected in the 1980""s, because of their chemical inertness and their inability to be metabolized, for use as artificial blood compounds. Given their high density and their complete insolubility in water based solutions, the only means for using these compounds as an artificial blood was to generate stable emulsions using hydrophobic phospholipids and standardizing the perfluorocarbon droplet size between 0.2-0.3 xcexcm. The research into these emulsions exploded with incomparable progress and in 1989, Flusol DA(copyright), was the first such emulsion approved by the FDA for clinical applications for use in Percutaneous Transluminary Angioplasty. Soon, other corporate products followed, such as Oxygent(copyright) from Alliance Pharmaceuticals. The first generation emulsions, although beneficial, also had some undesirable hemodilution effects. Through the vast academic and corporate research performed with the perfluorocarbons, it was found that higher weight/volume concentrations in PFC emulsions reduced the incidence of undesired effects and improved oxygen delivery and retention properties. This second generation of emulsions had a far greater impact. In the process, the critical properties of perfluorinated hydrocarbons and derivatives were carefully documented with an amazing lack of disparity from one research group to the next.
The oxygen carrying abilities of perfluorinated compounds stem from and are directly proportional to the amount of fluorine atoms within their structure. Similarly, the oxygen content of a given emulsion is directly proportional to the environmental pO2 of the emulsion and the weight percentage of the PFC in the overall volume of the emulsion. Moreover, perfluorocarbons have a kinetics of oxygen uptake and off-load that is twice as fast as the body""s normal oxygen delivery system, hemoglobin. These properties are further enhanced by the delivery of high concentrations of oxygen to patients receiving PFC emulsions.
In a study of patients receiving PFC emulsions IV, beneficial increases in pO2 were seen with every possible combination of gas and emulsion. With a low weight percentage emulsion (20% w/v) and room air, patients had a mean increase in pO2 from 82 mm Hg to 101 mm Hg. When inspiring pure oxygen, the values went from 291 mm Hg to 361 mm Hg (32). By the same token, marked increases in tissue pO2 were observed in animals following the administration of PFC emulsions. The most interesting aspect of these increases is that they persisted for several days and even when the levels of blood PFC concentrations could have been nearing zero.
Besides the studies of PFC emulsions for intravenous use as artificial blood, numerous groups have used the compounds in studies concerning reperfusion injury in harvested organs resulting from hypoxia and the preservation of physiological tissue oxygenation during transplant surgery (38-49). Summarily, the findings of these groups is that fluorocarbon emulsions or compounds have tremendous preservation effects on harvested tissue. Three such studies of particular interest are those of Kuroda et al. regarding the two layer method of pancreas preservation (50), Tanioka et al. regarding the use of the two layer method to improve islet isolation after long ischemic times (51), and the group of Urushihara et al. regarding the use of PFC""s and PFC emulsions in the cold preservation of rat pancreata (52). In the first study, dog pancreata were harvested and used. This study clearly demonstrated that a simple bilayer of perfluorocarbon, which being so dense and insoluble it coalesces at the bottom of a container, with another preservation medium significantly improves the function of cold-preserved panreata, to the point of comparable graft success with pancreata immediately transplanted after harvesting.
Urushihara et al also found similarly regarding preservation by using rat pancreata. In this study, however, the design was to compare the already proven preservation capabilities of PFC""s vs. PFC emulsions. In Urushihara, experimental design contained four groups. The first group consisted of pancreata perfused with a FC emulsion and then immersed in the emulsion while gassed with 95%O2/5%CO2 mixture. The second group differs from the first group only in that 100% N2 was used for gas bubbling. The third group was composed of pancreata perfused with liquid PFC, further immersed in PFC and then gassed, again with 95%O2/5%CO2. The fourth and final group is the same as the third group with the exception of using 100% N2 as the bubbling gas. The function of the organs after culture time was then assessed by graft transplantation (syngeneic) under the following criteria for a successful graft: if the graft reduced STZ induced hyperglycemia to levels below 200 mg/dL within 24 hours, and if this level was maintained for greater than two weeks, then a graft was deemed functional. Urushihara found that there was preservation in all groups for 24 hours, but at 48 hours all but group 3 had 0% graft success. Group 3, liquid PFC with 95%O2/5%CO2, was markedly superior to all the other groups in every aspect of preservation. Of the organs in the 24-hour culture group, 100% of the grafts transplanted were functional. Of the 48 hour culture group, 80% of the grafts transplanted (⅘) were functional. When compared to the 12-16 hour preservation times afforded by the standard organ procurement solutions, such as UW solution, PFC shows a distinct advantage. In fact, a group at the University of Washington is now using a two layer PFC method for perfusing pancreata after harvest and have seen substantial improvements in preservation time.
Of particular importance to our field of study, the group of Tanioka et al. applied the two-layer method to pancreatic islet cell harvesting. Their study was divided into five groups: The first group of dog pancreata were harvested and preserved for three hours using a bilayer of UW solution and PFC. At the end of this period, the Islets of Langerhans were isolated from the pancreata using the Ricordi method of human islet isolation modified for rat pancreata (53). The second group employed the same preservation method as the first, but for a period of 24 hours, at which time the islets were isolated. The third and fourth groups used just UW solution as a preservation media. In all of the first four groups, 95%O2/5%CO2 was bubbled in the preservation containers. The final group was an immediate harvest and autologous transplant group, as a control.
After the preservation periods and islet isolations in Tanioka, the islets were counted (pre-purification on a Ficoll gradient and post-purification), and transplanted, through cannulation of a mesenteric vein, into the liver. As in previous whole pancreas studies, graft success was determined by the reversal of hyperglycemia (glucose values below 200 mg/dL) in the pancreatectomized autologous donors, and by the maintenance of this reversal for a significant period of time. In the control group, the percentage recovery of the islets (post-purification count/pre-purification count) was 65.4% with 5000 IEQ""s/g of pancreas recovered. The experimental groups at three hours had values of 63.3% for the bilayer PFC group (5600 IEQ""s/g of pancreas) and 59.3% for the UW group (4700 IEQ""s/g of pancreas). Values at 24 hours of cold preservation for the two groups were 56.4% for the bi-layer PFC group (4000 IEQ""s/g of pancreas) and 39.3% for the UW group (1300 IEQ""s/g of pancreas). In the cell counts alone, the advantage of the use of PFC""s in the preservation prior to isolation was apparent. Further evidence for the superiority of the two-layer method in organ, and even single cell preservation, were the graft success rates. The control group had a graft success rate of 89% ({fraction (8/9)}) transplants. The three hour experimental groups had rates of 83% (⅚, bi-layer PFC group) and 33% ({fraction (2/6)}, UW group). The experimental 24 hour groups had rates of 56% ({fraction (5/9)}, bi-layer PFC group) and 0% ({fraction (0/9)}, UW group). This overwhelming evidence suggests that not only do perfluorocompounds act as a beneficial preservation media in whole organ harvesting, but also can be used in the preservation of isolated cells, such as the islets of Langerhans.
The arguments for the use of perfluroemulsions and perfluorocompounds in the area of cell and tissue preservation are sound. These findings established the background for our invention that is now described.
Therefore, the invention, even more broadly, relates to:
A composition comprising at least one glycosaminoglycan, at least one alginate and at least one perfluorocarbon substance, e.g., at least one emulsified perfluorocarbon substance to increase oxygen availability for encapsulated and/or immunoisolated cells and tissues, wherein:
the at least one glycosaminoglycan and/or the alginate are cross-linked or polymerized, e.g., the alginate is cross-linked or polymerized, for instance by addition of an inorganic salt, such as a calcium salt; or
the at least one glycosaminoglycan and the alginate are covalently bound, e.g., by means of a coupling reaction involving a linker molecule such as DVS; or
the at least one glycosaminoglycan and/or the alginate are cross-linked or polymerized, e.g., the alginate is cross-linked or polymerized, for instance by addition of an inorganic salt, such as a calcium salt, and the at least one glycosaminoglycan and the alginate are covalently bound, e.g., by means of a coupling reaction involving a linker molecule such as DVS, and the covalent binding can have been performed prior to cross-linking or polymerizing or vice versa;
Adding to or using with Biodritin an emulsified perfluorocarbon substance to increase oxygen availability to encapsulated and/or immunoisolated cells and tissues;
Gels comprising the composition; mixtures of such gels or of at least one such gel and at least one such composition;
Methods for making such compositions and gels;
Methods for using such compositions and gels, including as xe2x80x9cpaintsxe2x80x9d, sprays, matrices, beads, microcapsules;
Products comprising the composition or gel, e.g., xe2x80x9cpaintsxe2x80x9d, sprays, matrices, beads, microcapsules; and
The perfluorocarbon substances including but not limited to perfluorotributylamine (FC-43), perfluorodecalin, perfluorooctyl bromide or bis-perfluorobutyl-ethene.
A Biodritin heteropolysaccharide solution is transformed into gel microcapsules by dripping the solution into a calcium chloride solution. A Biodritin heteropolysaccharide solution is fashioned into a slab of any desired shape by contacting the Biodritin heteropolysaccharide and calcium chloride solution and thus gelling the Biodritin in the forms of the desired shape, such that the shape of the gel may be varied, as desired. The gel microcapsules or slab can have pancreas islets of Langerhans therein.
Biodritin can also be formed into a xe2x80x9cspaghettixe2x80x9d-like structure, prepared by extrusion of a Biodritin heteropolysaccharide solution over a line, e.g., through a cylindrical catheter tube containing inside a cotton or surgical line. The Biodritin solution is extruded together with the line into a calcium ion containing solution, whereby a gel instantly forms that contains the line. The gel is then matured in a calcium ion solution. One can form a protective outer layer, e.g., of poly-lysine/Biodritin, over the Biodritin spaghetti, to provide limited permeability to the device. The structuring of the spaghetti device is by the line in the interior and extending further out therefrom towards the surrounding gel cylinder, e.g., by the lateral extensions of the line, or by those provoked on the surface of the line by scraping it, e.g., with a knife blade.
A Biodritin spaghetti string can be used for implantation of cells or tissues therein contained, e.g., from the lateral extensions; or more than one Biodritin spaghetti strings may be used by being tied up together at each end. The strings can be further inserted within a biocompatible material such that the Biodritin spaghetti strings are protected from mechanical strains after implantation. And, these string devices can be used as a means of implanting cells or tissues in humans and animals for disease prevention or treatment, as is the case of islets of Langerhans implants for diabetes treatment.
The skilled artisan can implant a composition containing cells, such as islet cells, without undue experimentation, taking into account typical factors such as the age, sex, condition etc. of the patient, and the rate of secretion of a desired expression product of the cells, e.g., insulin (see also U.S. Ser. No. 08/417,652, filed Apr. 5, 1995, now U.S. Pat. No. 5,808,050 issued Sep. 15, 1998 incorporated herein by reference).
Thus, the invention relates to these products and methods for preparing and using them for encapsulating cells and tissues in a long term culture, transportation and/or transplantation in a medical treatment.
Analogous methods can be used to prepare products from Biodritin polymer networks; and, the invention accordingly relates to these products and methods for preparing and using them.
With respect to glycosaminoglycans, such as heparin sulfate, heparin and hyaluronic acid, these are not preferred for the invention as they can have defined cell-binding properties. With respect to material having defined cell binding properties which renders them unsuitable for use in the instant invention, hyaluronic acid binds specifically to CD-44, also known as xe2x80x9clymphocyte homing receptorxe2x80x9d (Hermes-antigen) or xe2x80x9cmajor hyaluronic acid receptor of mammalian cellsxe2x80x9d (Aruffo et al, 1990). CD-44 is expressed on the surfaces of most mammalian cells, rodent and primate hematopoietic cell types, fibroblastoid, neural and muscle cells (Rosenmann and St. John, 1993). Hyaluronic acid also binds to Versican, a large proteoglycan secreted by fibroblasts that promotes cell adhesion through interactions with components of the extracellular matrix and cell surface glycoproteins (Zimmermann, 1993).
Further, as to the use of materials having undesirable defined cell binding properties, heparin is also not preferred to be linked to alginate in the composition due to its well-known inhibition of the blood clotting mechanism. Heparan sulfate is also not preferred on the basis of its participation in cell-cell adhesion mechanisms. Heparan sulfate is linked to a membrane-bound proteoglycan that binds NCAM (neural cell adhesion molecule), thereby promoting homophilic cell adhesion (Cole et al, 1986). The Heparan sulfate binding domain of fibronectin is responsible for the binding of neurons, lymphocytes and other cell types to fibronectin, in the process of cell-cell adhesion (Liao et al, 1988). Further, heparan sulfate proteoglycans found on cell surfaces and in the extracellular matrix are binding sites for the basic fibroblast growth factor (bFGF) (Moscatelli et al, 1988).
Thus, having established that CIS is the most desirable glycosaminoglycan to utilize for the purposes of the present invention, the inventive heteropolysaccharides arise from stable, covalent chemical bonds between the structures of the at least one alginic acid salt, e.g., sodium alginate and the at least one glycosaminoglycan, e.g., CIS, for example, by a coupling reaction with divinyl sulfone, DVS. These reactants can produce a novel neo-hetero-polysaccharide conjugate having desired properties for use in cell and tissue biology, whenever biocompatibility and/or immunoisolation are critical issues. Along the same line, a semi-interpenetrating polymer network can be prepared by mixing CIS and alginate, at desired concentrations and forming a gel by addition of calcium ions that has similar properties to and benefits of the inventive heteropolysaccharides.
Thus, the invention can relate at least to two processes.
First, the present invention provides a process for preparing a heteropolysaccharide comprising covalently bonding at least one glycosaminoglycan, e.g., CIS, and at least one alginic acid salt, e.g., sodium alginate, preferably by a coupling reaction with a linker molecule, e.g., DVS, and an emulsified perfluorocarbon substance, including but not limited to perfluorotributylamine (FC-43), perfluorodecalin, perfluorooctyl bromide or bis-perfluorobutyl-ethene, such that the calcium binding sites in alginate are preserved and/or conserved during the coupling reaction. These sites provide the flexibility of using the heteropolysaccharide as a sol or a gel of desired strength, or as a composition having adjustable viscosity; and, the present invention relates such products from the heteropolysaccharide and to method for making and using them, e.g., by contacting with ions, such as calcium ions to prepare a gel, for instance, an infinite gel.
The second process forms a physical gel, e.g., of the semi-interpenetrating polymer network type, by addition of a polymerizing or cross-linking agent, e.g., calcium ions to a solution containing both at least one alginate, at least one glycosaminoglycan, e.g., chondroitin sulfate-4 and/or -6, and an emulsified perfluorocarbon substance, including but not limited to perfluorotributylamine (FC-43), perfluorodecalin, perfluorooctyl bromide or bis-perfluorobutyl-ethene. This gel can be a composition that can be varied to suit specific applications. The inventive s-IPN gel is stable and is formed by the complexation of calcium ions with poly-guluronic blocks of alginate to form the xe2x80x9cegg-boxxe2x80x9d structures that stabilize the gel.
Still, further within the principles just delineated, another formulation can be prepared by suitable combination of the formulations described above, in which a given amount of the covalently bound heteropolysaccharide conjugate is mixed in solution with alginate and CIS, to which solution calcium ions are added to form the gel.
Likewise, another formulation can be prepared by subjecting an inventive gel to a linking reaction so as to covalently bond glycosaminoglycan and alginate units to each other, e.g., by subjecting the gel to a coupling reaction involving a linker molecule such as DVS.
The invention further relates to uses of gels and compositions which can be formed into gels, especially biocompatible gels, such as in xe2x80x9cpaintsxe2x80x9d, sprays, matrices, beads, microcapsules, and the like, including novel uses such as xe2x80x9cspaghettixe2x80x9d-like material comprising a spine, e.g., a suture or thread, having a desired material connected thereto, e.g., cells connected to the spine, and an inventive gel surrounding the spine, e.g., in a cylindrical manner.
Documents are cited in this disclosure with a full citation for each appearing thereat and/or in a Reference List. These documents relate to the state-of-the-art to which this invention pertains, and each document cited herein and each document referenced or cited in a herein-cited documents are hereby incorporated by reference.
Cells and tissues may be immobilized and immunoisolated by three basic techniques: in extravascular chambers isolated but in the path of the blood stream, in spherical dispersions or microcapsules, and within macrocapsules. Using these techniques a great variety of cells and tissues of different animals have been immunoisolated and implanted in animals for development of therapeutic systems (reviewed by Christenson et al (1993)). Indeed, future applications of immunoisolated cell therapy are envisaged for diseases or conditions such as diabetes, hemophilia, hepatic failure, Alzheimer""s, Parkinson""s and Huntington""s diseases, affective disorders, hepatic failure and fertility problems (Christenson et al, 1993). A complete review of the questions involved in encapsulation and immunoisolation is presented in a recent volume edited by Goosen (1993).
In the case of diabetes mellitus, alternatives to daily insulin injections have been searched for control of type I diabetes, or insulin-dependent diabetes mellitus (IDDM). These included pancreas transplants, pumps to deliver insulin under a controlled program and, more recently, Langerhans"" islets transplantation. A review of sustained-release implants for insulin delivery has been published (Wang, 1991).
Several researchers have proposed different approaches to protect islet tissue from host attack after transplantation; these include encapsulation of islets in different materials such that insulin may be secreted but the beta cells in the islet tissue will be immunologically isolated from the host. Polysaccharides have been proposed to form membranes, as is the case of agarose, by Howell et al (1982) or alginate, by Tze and Tai (1982). Materials used include synthetic poly-acids and poly-bases, gelatin and polyamino acids (Young et al, 1989) as well as different polysaccharides: chemically modified dextran, to form poly-ionically bonded capsules (Lim and Hall, 1988, PCT Int. Appl. WO 8,800,327), entrapment in alginate followed by stabilization with poly-lysine and alginate (Chang and Wong, 1992, U.S. Pat. No. 5,084,350), as well as a combination of chitosan and carboxy-methyl cellulose to form capsules of controlled permeability (Shioya and Hirano, 1990, U.S. Pat. No. 5,089,272).
Additionally, recent work bearing on regeneration of skin in culture has pointed out the important role of GAG""s in the process, in studies where mixtures of collagen and GAG were used as support (Murphy et al, 1990; Yannas et al, 1990). Along the same line, keratinocytes and fibroblasts grown on a nylon mesh produced a dermal-like matrix containing proteoglycans (Slovakia et al, 1993).
The importance of the extra-cellular matrix components for the normal development of the skin system lends support to a basis of the instant invention, e.g., that foreign molecules combined or structured with specified GAG""s, e.g., preferably CIS in the case of this invention, will constitute ideal protecting materials in transplantation or implantation of cells or tissues of human or animal origin, with the purpose of treating or controlling disease.
However, the art heretofore fails to teach or suggest the particular heteropolysaccharides polymer networks containing additionally an emulsified perfluorocarbon substance which is capable of increasing oxygen availability for encapsulated and/or immunoisolated cells and tissues, thus, prolonging the vitality of the encapsulated cells and tissues for transportation and/or transplantation, and products therefrom and processes and uses of the invention.
Further, essential constituents of the extra-cellular matrix in connective tissues, of cell membranes and endothelial lining, and the overall presence of GAG""s demonstrates their importance in matrix formation and extension, and in cell-matrix and cell-cell interactions. Although GAG""s occur in an organism mostly linked to proteins, as proteoglycans, it has been demonstrated that only the protein portion is immunogenic; the glycosaminoglycan, e.g., CIS component, is not immunogenic by itself (Hirschmann and Dziewiatkowski, 1966; Loewi and Muir, 1965).
However, use of a GAG with an alginate salt, for instance GAG-alginate biomolecules, e.g., via a coupling reaction with a linker molecule, to form a heteropolysaccharide conjugate and products therefrom and processes and uses of the invention are not heretofore taught or suggested. Moreover, the formation of a polymer network, e.g., of a semi-interpenetrating polymer network, based on the two components, alginate and GAG, e.g., CIS, rendered in gel form by addition of ions such as inorganic ions, e.g., calcium ions, has not been taught nor suggested in the prior art. Nor has a polymer network comprising a heteropolysaccharide from coupling GAG-alginate present in a GAG-alginate polymer network, been heretofore taught or suggested.
In the case of diabetes, the depth of interest in discovering the best way to use islets in transplantation is demonstrated by two recently published papers, one dealing with storage and preservation of islets (Jindal and Gray, 1994) and the other with the action of prednisone on the islet autograft function (Rodrigues Rilo et al, 1994).
U.S. Pat. No. 4,409,331 to Lim relates to the encapsulation of islets in polymeric material formed from alginate and poly-lysine; Lim and Sun (1980) discussed the microencapsulation of islets to form a bioartificial pancreas. Chitosan microspheres were developed that bind to GAG receptors on cell surfaces (Gallo et al U.S. Pat. No. 5,129,877). Collagen-GAG microcapsules were proposed as drug delivery systems, to deliver anti-microbial agents (Rase et al U.S. Pat. No. 5,169,631). Polyacrylates were also developed as encapsulation materials and have also been co-polymerized with alginate, as discussed by Stevenson and Sefton (1993). However, use of GAG having an emulsified perfluorocarbon substance with an alginate salt, GAG-alginate biomolecules, e.g., in physical mixture or bound via a coupling reaction with a linker molecule, to form a heteropolysaccharide or a physical network, e.g., s-IPN, gel and products therefrom and processes and uses of the invention, are not taught or suggested.
It is emphasized, however, that none of the art heretofore has GAG containing a perfluorinated substance acted as the major biocompatibility agent between a foreign chemical structure or device and host organism, especially in a composition with an alginate.
The synthesis and properties of glycoconjugates has been reviewed in a book edited by Lee and Lee (1994). The new class of structures now being described herein belongs to a group of glycopolymers formed by joining together two different natural heteropolysaccharides by reaction with an unnatural tether e.g., divinyl sulfone. This new class would enter the list of neoglycoconjugates compiled by Magnusson et al (1994) under the heading xe2x80x9cglycopolymers with gel forming propertiesxe2x80x9d.
On the other hand, the physical gel formed by adding calcium ions to solutions containing variable concentrations of GAG, e.g., CIS, and alginate belongs to the group of polymers known as semi-interpenetrating polymer networks, recently discussed by LaPorte (1997). In this category, one type of polymer is enmeshed and entrapped by a second polymer, which is cross-linked to stabilize the structure.
However, that compositions of the invention may be characterized as xe2x80x9cglycopolymers with gel forming propertiesxe2x80x9d or as a xe2x80x9cpolymer networkxe2x80x9d, e.g., xe2x80x9ca semi-interpenetrating polymer networkxe2x80x9d does not mean that the invention has heretofore been taught or suggested.
Thus, the present invention provides a novel hetero-polysaccharide conjugate or complex, gels and/or sols derived therefrom, and to the synthesis, purification and utilization of a novel glycopolymer and/or heteropolysaccharide and/or gels with a perfluorinated substance, solutions and/or sols, herein referred to as Biodritin composition. The inventive hetero-polysaccharide is preferably formed from covalent bonding between at least one glycosaminoglycan, e.g., chondroitin sulfate-4 and/or -6, and at least one alginic acid salt, e.g., such that a gel and/or sol is formed, e.g., and a perfluorinated substance such as an emulsified perfluorocarbon substance preferably but not limited to perfluorotributylamine (FC-43), perfluorodecalin, perfluorooctyl bromide or bis-perfluorobutyl-ethene, in the presence of an appropriate counterion, e.g., calcium.
An additional formulation of the invention combines the two polysaccharides, alginate and GAG, e.g., CIS, in aqueous solutions having variable concentrations of either component, through the formation of a gel of the type semi-interpenetrating polymer network, by addition of a cross-linking or polymerizing agent, e.g., calcium ions. A third formulation combines the two concepts in the same preparation, in suitable proportion, which can be established by the skilled artisan, without undue experimentation, from this disclosure and the knowledge in the art.
Therefore, the invention, provides:
a composition comprising at least one glycosaminoglycan, e.g., CIS, an emulsified perfluorocarbon substance, and at least one alginate, e.g., sodium alginate, wherein:
the at least one glycosaminoglycan and/or the alginate are cross-linked or polymerized, e.g., the alginate is cross-linked or polymerized, for instance by addition of an inorganic salt, such as a calcium salt; or
the at least one glycosaminoglycan and the alginate are covalently bound, e.g., by means of a coupling reaction involving a linker molecule such as DVS; or
the at least one glycosaminoglycan and/or the alginate are cross-linked or polymerized, e.g., the alginate is cross-linked or polymerized, for instance by addition of an inorganic salt, such as a calcium salt, and the at least one glycosaminoglycan and the alginate are covalently bound, e.g., by means of a coupling reaction involving a linker molecule such as DVS, and the covalent binding can have been performed prior to cross-linking or polymerizing or vice versa; and,
having a perfluorinated substance in the Biodritin polymer formulation, the perfluorinated substance is preferably an emulsified perfluorocarbon substance, preferably but not limited to perfluorotributylamine (FC-43), perfluorodecalin, perfluorooctyl bromide or bis-perfluorobutyl-ethene,
gels comprising the composition; mixtures of such gels or of at least one such gel and at least one such composition; and,
methods for making such compositions and gels; and
methods for using such compositions and gels, including as xe2x80x9cpaintsxe2x80x9d, sprays, matrices, beads, microcapsules; and,
products comprising the composition or gel, e.g., xe2x80x9cpaintsxe2x80x9d, sprays, matrices, beads, and microcapsules.
The inventive composition can be obtained by admixing 0.5% to 5.0%, e.g., 1% to 3%, by weight or by volume of alginate, e.g., sodium alginate, and about 1.0%, e.g., 1.5% or 2.0%, to up to 20% to 30%, e.g., 25%, by weight or by volume of GAG, e.g., CIS. That is, in general, in forming inventive compositions the alginate to GAG ratio can be 0.5:30; but, preferably three (3) to four (4) times more GAG is present than alginate (by weight or by volume), i.e., a preferred alginate to GAG ratio in forming inventive compositions is 1:3 to 1:4. Alginates come in varying viscosities, e.g., high viscosity, medium viscosity. Medium viscosity alginates are preferred as high viscosity alginates can result in harder beads. Further, the amount of alginate employed in forming an inventive composition can be limited by the viscosity of the alginate; with a medium viscosity alginate, 5% by weight or volume alginate is rather viscous. Furthermore, the skilled artisan can readily appreciate that the volume and viscosity or hardness of compositions, gels and products therefrom, e.g., beads, etc. can be varied without undue experimentation by varying the amount of GAG, e.g., CIS and alginate, e.g., sodium alginate. For instance, when a softer composition, gel or product, having more volume, is desired, more GAG is employed, i.e., the amount of GAG by weight or volume is increased; and, when physical resistance, hardness, and like properties are desired, more alginate is employed, i.e., the amount of alginate by weight or volume is increased.
When a linker molecule is used in forming an inventive compound, it can be used in an amount relative to the amount of alginate, or to the amount of GAG present, e.g., an amount from 50% of to equal by weight, volume or stoichiometrically to the amount of alginate present, or to the amount of GAG present, or twice or thrice the weight, volume or stoichiometric amount of alginate or GAG present. When an inorganic ion is used as a cross-linking or polymerizing agent in forming a composition of the invention, the inorganic ion is preferably a calcium ion, e.g., calcium chloride, which can be used in an amount relative to the amount of alginate present, e.g., in an amount of about 0.05% to 2.0% by weight or by volume, e.g., about 1.0% by weight or by volume.
Gels can be essentially covalent, or part covalent and part interpenetrating. From analysis of inventive gels, e.g., HPLC analysis showing bands corresponding to alginate, CIS and a covalent structure, it is believed that gels of the invention form interpenetrating networks. However, Applicants do not necessarily wish to be bound by any one particular theory.
Biodritin containing an emulsified perfluorocarbon substance can be applied to surgical wounds or sutures, to protect from adherences; it also serves as a matrix material for supporting cells for culturing or other applications, particularly, in which cells must be maintained with high vitality and suspended in a non-aggregated state.
An object of the invention is to provide a composition capable of supporting cells for culturing, transportation and/or transplantation wherein the cells can be maintained in vitro long term and with high vitality and low mortality rate by having a composition capable of enhancing oxygen diffusion.
In this disclosure, xe2x80x9ccomprisesxe2x80x9d, xe2x80x9ccomprisingxe2x80x9d and the like can have the meaning ascribed to them in U.S. Patent Law and can mean xe2x80x9cincludesxe2x80x9d, xe2x80x9cincludingxe2x80x9d and the like.
These and other embodiments will be described and/or will be obvious from the following detailed description.